Acetoin is a popular food flavoring that is widely used in the world as a component of flavorings of cream, yogurt, strawberry and so on. With a pleasant buttery odor, acetoin is often used to enhance the flavor of cream, cheese, coffee, nut, etc. Acetoin can also change the flavor of beer and cheese during fermentation. Nowadays as the consumption of dairy products continues to grow, more and more people enjoy foods with a cream flavor. Research and development in acetoin production have drawn attention of companies and research institutions throughout the world.
At present, methods for acetoin production in the laboratory mainly include the following: extracting acetoin from acetoin-containing plants; biological methods; oxidizing 2,3-butanedione using catalysts; oxidizing butanone using electrochemical methods; hydrolyzing linear ketones in sulfuric acid dilution using thallium salt; and synthesizing acetoin from butanedione or 2,3-butanediol.
Studies on acetoin production were first reported in the early twentieth century. One method employed was partial deoxidation of 2,3-butanedione using zinc and acids. Another method was selective oxidation of 2,3-butanediol. Recently, many biological techniques for acetoin production have been reported, for example, converting 2,3-butanediol into acetoin with mycoderma, or using aspergillus, penicillium or other epiphytes to act on sugarcane juice. But these studies were conducted in laboratory settings. To meet the needs for environmental protection and green technologies, biological methods will be the major direction for future research in acetoin production.
The industrial methods for acetoin production are mainly chemosynthesis using 2,3-butanedione as the substrate. In 1989, Ehime University in Japan successfully obtained acetoin by reducing 2,3-butanedione in the system of Zn—ZnCl-EtOH. In this method, the reaction was carried out with heating and stirring at about 70° C.˜80° C. at the natural pressure. Acetoin was obtained after further separation and purification, resulting in a 71% recovery. In 1992, Hangzhou University in China developed a new method to produce acetoin by reduction with NaHSe. In this method, selenium powder was added into a NaHB solution in a stirring reactor. After NaHSe was formed under vacuum, a mixed solution of acetic acid and ethanol, and 2,3-butanedione dissolved in tetrahydrofuran were added into the stirring reactor, where the reaction was carried out at the room temperature. The yield of this method was 57%.
In 1998, Martin Studer et al. of Witwatersrand University used platinum denaturalized by 10,11-dihydrgen cinchona ledgeriana (HCD) as a catalyst to selectively deoxidize 2,3-butanedione by hydrogenation. In this method, butanedione, catalyst and HOD in toluene were added into a high-pressure reactor. The reaction pressure was 10.7 Mpa, while the reaction temperature was 0° C.˜25° C. The reaction was stopped after about 10 min. The yield of the method was 85%, and some optically active byproducts were also obtained. Since this was a catalytic hydrogenation reaction, controlling the reaction conditions was critical. If the reaction continued, acetoin would be further converted into 2,3-butanediol, and the recovery rate of the acetoin product would only reach 50%. Slipszenko et al. of Hull University also conducted research on butanedione deoxidation by selective catalytic hydrogenation using platinum as the catalyst. But the solvent they used was methylene dichloride, and the reaction pressure, temperature, and yield were 1 Mpa, 5° C.˜25° C., and 85%, respectively. In this method, more (R)-acetoin enantiomer could be produced by controlling the reaction time and the hydrogen pressure, and the yield could reach 70%.
Since catalytic hydrogenation is carried out at a high pressure, specific equipments are required. In addition, the catalyst used in the reaction is an expensive precious metal. Problems concerning the catalyst, such as manufacturing, denaturalization, regeneration and metal poisoning, have not been solved and thus confine the method to laboratory studies.
In 1992, Hummel et al. in the United States used enzymes from microorganisms as catalysts for acetoin production. In this method, butanedione reductase is isolated from lactic bacteria or yeast Saccharomycetes, and used to convert butanedione to acetoin in the presence of NADPH at pH 5 and 70° C. The yield of the reaction can reach as high as 100%. Because the enzymes act as stereospecific catalysts, this method produced chiral compounds, generating no or few enantiomers. The advantages of reductases, which are highly selective, high-yield, and safe in food additive production, are obvious. But the key step of this method is to obtain butanedione reductases needed for the reactions. Enzyme-based methods remain a very important research area in the era of green technologies.
Microbial fermentation is an important biological method for acetoin production. The metabolic pathway of acetoin production using glucose or other substrates has been elaborated (FIG. 1), which provides the theoretical basis for fermentative production of acetoin. Although there were some reports including a few patents on this method, most of them were still restricted to laboratory studies. Isolating a high-yield, acetoin-producing bacterial strain is important for fermentative acetoin production. So far, the following strains for acetoin production have been reported: Klebsiella pneumoniae, Klebsiella oxytoca, Aeromonas hydrophilia, Bacillus subtilis, Bacillus polymyxa, Bacillus licheniformis, Serratia marcescens, Listeria monocytogenes, Aerobacter aerogenes, Bacillus amyloliquefaciens, Enterobacter aerogenes, Lactococcus lactis, Lactobacillus casei, Streptococcus thermophilus, Leuconoctoc mesenteroides, Leuconoctoc lactis, Leuconoctoc oenos, Leuconoctoc pseudomesenteroides, Bacillus stearothermophilus, Hanseniaspora guillieromondil, Saccharomyces carlsbergensis, Saccharomycodes ludwigii, Zygosaccharomyces bailli, Zygosaccharomyces fermentati, and so on. But all of these strains share the problem that the yield of acetoin is too low, or acetoin is produced only as a by-product of 2,3-butanediol biosynthesis. Therefore, it is difficult to use the strains above for industrial-scale production of acetoin.
Due to the low concentration of acetoin in plants, extracting acetoin from plant materials is costly and not suitable for commercialization. While chemical synthesis can produce high yield acetoin, extreme reaction conditions and sophisticated equipments are required. Moreover, the resulting acetoin is not a natural product and there are serious concerns with environmental protection. The methods of biosynthesis, including microbial fermentation, have been only studied in the laboratories mostly because of low yield of the product, which is caused by problems in the strain used, enzyme activity, optimization of fermentation conditions, or process controls.